Evaluation of dextromethorphan metabolism using hepatocytes from CYP2D6 poor and extensive metabolizers

It is important to estimate the defective metabolism caused by genetic polymorphism of drug metabolizing enzymes before the clinical stage. We evaluated the utility of cryopreserved human hepatocytes of CYP2D6 poor metabolizer (PM) for the estimation of the metabolism in PM using dextromethorphan (DEX) as the probe drug for CYP2D6 substrate. The results of low formations of dextrorphan (DXO) and 3-hydroxymorphinan (3-HM) in CYP2D6 PM hepatocytes incubated with dextromethorphan reflected the clinical data. Formation of 3-methoxymorphinan (3-MEM) normalized by CYP3A4/5 activity in the PM hepatocytes reached about 2.8-fold higher than that in CYP2D6 extensive metabolizer (EM) hepatocytes, which clearly showed the compensatory metabolic pathway of O-demethylation catalyzed by CYP2D6 as seen in clinical study. On the contrary, in the condition of the EM hepatocytes with CYP2D6 inhibitors, the enhancement of 3-MEM formation was not observed. In phase II reaction, the glucuronide formation rate of DXO in the PM hepatocytes was lower than that in the EM hepatocytes, which was consistent with clinical data of DXO-glucuronide (DXO-glu) concentration. These results would suggest that CYP2D6 PM hepatocytes could be a good in vitro tool for estimating CYP2D6 PM pharmacokinetics.     

Change from the CYP2D6 extensive metabolizer to the poor metabolizer phenotype during treatment With bupropion

Some data indicate that bupropion inhibits the cytochrome P-450 enzyme CYP2D6, but very little published data is available on the extent of this inhibition. The objective of the present study was to quantify this inhibition in a subject treated with bupropion for smoking cessation. Genotypically, the patient was a CYP2D6 homozygous extensive metabolizer (EM). His CYP2D6 phenotype was assessed using the test drug dextromethorphan before, during, and after treatment with bupropion. During treatment with bupropion, he clearly changed from the EM to the poor metabolizer (PM) phenotype. Although the results from a single patient should be interpreted with great caution, the extent of the interaction indicates that bupropion might be a CYP2D6 inhibitor as potent as the most powerful CYP2D6 inhibitors known, such as quinidine and paroxetine.     

CYP3A4 mediates dextropropoxyphene N-demethylation to nordextropropoxyphene: human in vitro and in vivo studies and lack of CYP2D6 involvement

The individual cytochrome P450 isoforms in dextropropoxyphene N-demethylation to nordextropropoxyphene were determined and the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in cytochrome P4502D6 (CYP2D6) extensive (EM) and poor (PM) subjects were characterized. Microsomes from six CYP2D6 extensive metabolizers and one CYP2D6 poor metabolizer were used with isoform specific chemical and antibody inhibitors and expressed recombinant CYP enzymes. Groups of three CYP2D6 EM and PM subjects received a single 65-mg oral dose of dextropropoxyphene, and blood and urine were collected for 168 and 96 h, respectively. Nordextropropoxyphene formation in vitro was not different between the CYP2D6 extensive metabolizers (Km = 179 +/- 74 microM, Cl(int) = 0.41 +/- 0.26 ml mg(-1)h(-1)) and the PM subject (K = 225 microM, Cl(int) = 0.19 ml mg(-1) h(-1)) and was catalysed predominantly by CYP3A4. There was no apparent difference in the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in CYP2D6 EM and PM subjects. CYP3A4 is the major CYP enzyme catalysing the major metabolic pathway of dextropropoxyphene metabolism. Hence variability in the pharmacodynamic effects of dextropropoxyphene are likely due to intersubject variability in hepatic CYP3A4 expression and/or drug-drug interactions. Reported CYP2D6 phenocopying is not due to dextropropoxyphene being a CYP2D6 substrate.     

Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes

1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.     

Effects of CYP2D6 status on harmaline metabolism, pharmacokinetics and pharmacodynamics, and a pharmacogenetics-based pharmacokinetic model

Harmaline is a β-carboline alkaloid showing neuroprotective and neurotoxic properties. Our recent studies have revealed an important role for cytochrome P450 2D6 (CYP2D6) in harmaline O-demethylation. This study, therefore, aimed to delineate the effects of CYP2D6 phenotype/genotype on harmaline metabolism, pharmacokinetics (PK) and pharmacodynamics (PD), and to develop a pharmacogenetics mechanism-based compartmental PK model. In vitro kinetic studies on metabolite formation in human CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) hepatocytes indicated that harmaline O-demethylase activity (V_max/K_m) was about 9-fold higher in EM hepatocytes. Substrate depletion showed mono-exponential decay trait, and estimated in vitro harmaline clearance (CL_int, μL/min/10⁶ cells) was significantly lower in PM hepatocytes (28.5) than EM hepatocytes (71.1). In vivo studies in CYP2D6-humanized and wild-type mouse models showed that wild-type mice were subjected to higher and longer exposure to harmaline (5 and 15 mg/kg; i.v. and i.p.), and more severe hypothermic responses. The PK/PD data were nicely described by our pharmacogenetics-based PK model involving the clearance of drug by CYP2D6 (CL_CYP2D6) and other mechanisms (CL_other), and an indirect response PD model, respectively. Wild-type mice were also more sensitive to harmaline in marble-burying tests, as manifested by significantly lower ED₅₀ and steeper Hill slope. These findings suggest that distinct CYP2D6 status may cause considerable variations in harmaline metabolism, PK and PD. In addition, the pharmacogenetics-based PK model may be extended to define PK difference caused by other polymorphic drug-metabolizing enzyme in different populations.     

Disposition and metabolic fate of atomoxetine hydrochloride: the role of CYP2D6 in human disposition and metabolism.

The role of the polymorphic cytochrome p450 2D6 (CYP2D6) in the pharmacokinetics of atomoxetine hydrochloride [(-)-N-methyl-gamma-(2-methylphenoxy)benzenepropanamine hydrochloride; LY139603] has been documented following both single and multiple doses of the drug. In this study, the influence of the CYP2D6 polymorphism on the overall disposition and metabolism of a 20-mg dose of (14)C-atomoxetine was evaluated in CYP2D6 extensive metabolizer (EM; n = 4) and poor metabolizer (PM; n = 3) subjects under steady-state conditions. Atomoxetine was well absorbed from the gastrointestinal tract and cleared primarily by metabolism with the preponderance of radioactivity being excreted into the urine. In EM subjects, the majority of the radioactive dose was excreted within 24 h, whereas in PM subjects the majority of the dose was excreted by 72 h. The biotransformation of atomoxetine was similar in all subjects undergoing aromatic ring hydroxylation, benzylic oxidation, and N-demethylation with no CYP2D6 phenotype-specific metabolites. The primary oxidative metabolite of atomoxetine was 4-hydroxyatomoxetine, which was subsequently conjugated forming 4-hydroxyatomoxetine-O-glucuronide. Due to the absence of CYP2D6 activity, the systemic exposure to radioactivity was prolonged in PM subjects (t(1/2) = 62 h) compared with EM subjects (t(1/2) = 18 h). In EM subjects, atomoxetine (t(1/2) = 5 h) and 4-hydroxyatomoxetine-O-glucuronide (t(1/2) = 7 h) were the principle circulating species, whereas atomoxetine (t(1/2) = 20 h) and N-desmethylatomoxetine (t(1/2) = 33 h) were the principle circulating species in PM subjects. Although differences were observed in the excretion and relative amounts of metabolites formed, the primary difference observed between EM and PM subjects was the rate at which atomoxetine was biotransformed to 4-hydroxyatomoxetine.     

Identification of cytochrome P-450 isoforms responsible for cis-tramadol metabolism in human liver microsomes.

The metabolism of cis-tramadol has been studied in human liver microsomes and in cDNA-expressed human cytochrome P-450 (CYP) isoforms. Human liver microsomes catalyzed the NADPH-dependent metabolism of tramadol to the two primary tramadol metabolites, namely, O-desmethyl-tramadol (metabolite M1) and N-desmethyl-tramadol (metabolite M2). In addition, tramadol was also metabolized to two minor secondary metabolites (each comprising < or =3.0% of total tramadol metabolism), namely, N,N-didesmethyl-tramadol (metabolite M3) and N,O-didesmethyl-tramadol (metabolite M5). Kinetic analysis revealed that multiple CYP enzymes were involved in the metabolism of tramadol to both M1 and M2. For the high-affinity enzymes involved in M1 and M2 formation, K(m) values were 116 and 1021 microM, respectively. Subsequent reaction phenotyping studies were performed with a tramadol substrate concentration of 250 microM. In studies with characterized human liver microsomal preparations, good correlations were observed between tramadol metabolism to M1 and M2 and enzymatic markers of CYP2D6 and CYP2B6, respectively. Tramadol was metabolized to M1 by cDNA-expressed CYP2D6 and to M2 by CYP2B6 and CYP3A4. Tramadol metabolism in human liver microsomes to M1 and M2 was markedly inhibited by the CYP2D6 inhibitor quinidine and the CYP3A4 inhibitor troleandomycin, respectively. In summary, this study demonstrates that cis-tramadol can be metabolized to tramadol metabolites M1, M2, M3, and M5 in human liver microsomal preparations. By kinetic analysis and the results of the reaction phenotyping studies, tramadol metabolism in human liver is catalyzed by multiple CYP isoforms. Hepatic CYP2D6 appears to be primarily responsible for M1 formation, whereas M2 formation is catalyzed by CYP2B6 and CYP3A4.     

CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes

AIM: To determine the Michaelis-Menten kinetics of hydrocodone metabolism to its O- and N-demethylated products, hydromorphone and norhydrocodone, to determine the individual cytochrome p450 enzymes involved, and to predict the in vivo hepatic intrinsic clearance of hydrocodone via these pathways. METHODS: Liver microsomes from six CYP2D6 extensive metabolizers (EM) and one CYP2D6 poor metabolizer (PM) were used to determine the kinetics of hydromorphone and norhydrocodone formation. Chemical and antibody inhibitors were used to identify the cytochrome p450 isoforms catalyzing these pathways. Expressed recombinant cytochrome p450 enzymes were used to characterize further the metabolism of hydrocodone. RESULTS: Hydromorphone formation in liver microsomes from CYP2D6 EMs was dependent on a high affinity enzyme (Km = 26 microm) contributing 95%, and to a lesser degree a low affinity enzyme (Km = 3.4 mm). In contrast, only a low affinity enzyme (Km = 8.5 mm) formed this metabolite in the liver from the CYP2D6 PM, with significantly decreased hydromorphone formation compared with the livers from the EMs. Norhydrocodone was formed by a single low affinity enzyme (Km = 5.1 mm) in livers from both CYP2D6 EM and PM. Recombinant CYP2D6 and CYP3A4 formed only hydromorphone and only norhydrocodone, respectively. Hydromorphone formation was inhibited by quinidine (a selective inhibitor of CYP2D6 activity), and monoclonal antibodies specific to CYP2D6. Troleandomycin, ketoconazole (both CYP3A4 inhibitors) and monoclonal antibodies specific for CYP3A4 inhibited norhydrocodone formation. Extrapolation of in vitro to in vivo data resulted in a predicted total hepatic clearance of 227 ml x h-1 x kg-1 and 124 ml x h-1 x kg-1 for CYP2D6 EM and PM, respectively. CONCLUSIONS: The O-demethylation of hydrocodone is predominantly catalyzed by CYP2D6 and to a lesser extent by an unknown low affinity cytochrome p450 enzyme. Norhydrocodone formation was attributed to CYP3A4. Comparison of recalculated published clearance data for hydrocodone, with those predicted in the present work, indicate that about 40% of the clearance of hydrocodone is via non-CYP pathways. Our data also suggest that the genetic polymorphisms of CYP2D6 may influence hydrocodone metabolism and its therapeutic efficacy.     

In vivo metabolism of the new designer drug 1-(4-methoxyphenyl)piperazine (MeOPP) in rat and identification of the human cytochrome P450 enzymes responsible for the major metabolic step

1. The in vivo metabolism of 1-(4-methoxyphenyl)piperazine (MeOPP), a novel designer drug, was studied in male Wistar rats. 2. MeOPP was mainly O-demethylated to 1-(4-hydroxyphenyl)piperazine (4-HO-PP) in addition to degradation of the piperazine moiety. 3. O-demethylation, the major metabolic step, was studied with cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes in pooled human liver microsomes (pHLM) and in single donor human liver microsomes with CYP2D6 poor metabolizer genotype (PM HLM). 4. CYP2D6 catalysed O-demethylation with apparent Km and Vmax values of 48.34 +/- 14.48 microM and 5.44 +/- 0.47 pmol min(-1) pmol(-1) CYP, respectively. pHLM catalysed the monitored reaction with an apparent Km = 204.80 +/- 51.81 microM and Vmax = 127.50 +/- 13.25 pmol min(-1) mg(-1) protein. 5. The CYP2D6-specific chemical inhibitor quinidine (1 and 3 microM) significantly inhibited 4-HO-PP formation by 71.9 +/- 4.8% and by 98.5% +/- 0.5%, respectively, in incubation mixtures with pHLM and 200 microM MeOPP. 6. O-demethylation was significantly lower in PM HLM compared with pHLM (70.6% +/- 7.2%). 7. These data suggest that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MeOPP O-demethylation.     

The role of CYP2C19 in the metabolism of (+/-) bufuralol, the prototypic substrate of CYP2D6.

Upon characterization of baculovirus-expressed cytochrome P-450 (CYP) 2C19, it was observed that this enzyme metabolized (+/-) bufuralol to 1'hydroxybufuralol, a reaction previously understood to be selectively catalyzed by CYP2D6. The apparent K(m) for this reaction was 36 microM with recombinant CYP2C19, approximately 7-fold higher than for recombinant CYP2D6. The intrinsic clearance for this reaction was 37-fold higher with CYP2D6 than for CYP2C19. The involvement of human CYP1A2 in bufuralol 1'-hydroxylation was also confirmed using the recombinant enzyme. Using S-mephenytoin as an inhibitor, the K(i) for inhibition of recombinant CYP2C19-mediated bufuralol hydroxylation was 42 microM, which is the approximate K(m) for recombinant CYP2C19-mediated S-mephenytoin metabolism. The classic CYP2D6 inhibitors quinidine and quinine showed no inhibition of CYP2C19-catalyzed bufuralol metabolism at concentrations that abolished CYP2D6-mediated bufuralol metabolism. Ticlopidine, a potent inhibitor of CYP2C19 and CYP2D6, inhibited bufuralol 1'-hydroxylation by each of these enzymes equipotently. In human liver microsomes that are known to be deficient in CYP2D6 activity, it was shown that in the presence of quinidine, the K(m) shifted from 14 to 38 microM. This is consistent with the K(m) determination for recombinant CYP2C19 of 36 microM. In human liver microsomes that have high CYP2D6 and CYP2C19 activity, the K(m) shifted to 145 microM in the presence of S-mephenytoin and quinidine, consistent with the K(m) determined for CYP1A2. This data suggests that bufuralol, and possibly other CYP2D6 substrates, have the potential to be metabolized by CYP2C19.     

Substrate specific metabolism by polymorphic cytochrome P450 2D6 alleles.

A comparative metabolism study was performed for bufuralol, dextromethorphan, imipramine, mianserin, sparteine, tamoxifen, haloperidol and two drug candidates (Rec27/0110 and Rec15/2739) on V79 cells genetically engineered to express human cytochrome P450 (CYP) variants 2D6*1, 2D*2, 2D*9 and 2D*17. Unexpectedly, the CYP2D6*17 dependent metabolism profile of haloperidol and Rec27/0110 were found to differ from all other substrates tested. Some of these known standard substrates are frequently applied in marker reactions for CYP2D6 and with these standard substrates, CYP2D6*1 is known to be the most active form. In both cases of haloperidol and Rec27/0110 the variant form CYP2D6*17 had equal or higher activity compared to the CYP2D6*1 form. Results obtained with the V79 cells were confirmed using microsomal preparation of yeast cells expressing the variants CYP2D6*1 and CYP2D6*17 and CYP2D6 inhibitor quinidine. In conclusion, there is no general rule for a variant dependent metabolism profile by cytochrome P450 2D6 indicating that the activity profile of the CYP2D6 alleles may be substrate specific, thus may be reflected in pharmacokinetics consequences for individuals.     

Identification of human cytochrome P450 2D6 as major enzyme involved in the O-demethylation of the designer drug p-methoxymethamphetamine.

p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.     

CYP2D6 is primarily responsible for the metabolism of clomiphene

Clomiphene is a first line treatment for anovulation, a common cause of infertility. Response to clomiphene is variable and unpredictable. Tamoxifen is structurally related to clomiphene, and also shows considerable variation in response. CYP2D6 and CYP3A4 are major contributors to the metabolism of tamoxifen. The aim of the present work was to define the role of CYP2D6 and CYP3A4 in the in vitro metabolism of enclomiphene, regarded by some as the more active isomer of clomiphene. Enclomiphene (25 microM) was incubated with human liver microsomes (from 4 extensive (EM) and 1 poor metaboliser with respect to CYP2D6) and with microsomes from lymphoblastoid cells expressing CYP2D6. Microsomes from all the EM livers and recombinant CYP2D6 metabolised enclomiphene (the disappearance of drug ranged from 40-60%). No metabolism was detected in microsomes from the PM liver. Quinidine (1 microM) completely inhibited the metabolism of enclomiphene by all the EM livers and by recombinant CYP2D6 (p<0.001, one way ANOVA). Ketoconazole (2 microM) had no significant effect on enclomiphene metabolism in 3 out of the 4 EM livers. The extent of enclomiphene metabolism was correlated with the amount of CYP2D6 present (p<0.001, Pearson correlation test). The findings indicate that CYP2D6 is primarily responsible for the metabolism of enclomiphene.     

Identification of cytochrome p450 enzymes involved in the metabolism of 4'-methyl-alpha-pyrrolidinopropiophenone, a novel scheduled designer drug, in human liver microsomes.

4'-Methyl-alpha-pyrrolidinopropiophenone (MPPP) is a new drug of abuse. It is believed to have an abuse potential similar to that of amphetamines. Previous studies with Wistar rats had shown that MPPP was metabolized mainly by hydroxylation in position 4' followed by dehydrogenation to the corresponding carboxylic acid. The aim of the study presented here was to identify the human hepatic cytochrome p450 (p450) enzymes involved in the biotransformation of MPPP to 4'-hydroxymethyl-pyrrolidinopropiophenone. Baculovirus-infected insect cell microsomes and human liver microsomes were used for this purpose. Only CYP2C19 and CYP2D6 catalyzed this hydroxylation. The apparent Km and Vmax values for the latter were 9.8 +/- 2.5 microM and 13.6 +/- 0.7 pmol/min/pmol p450, respectively. CYP2C19 was not saturable over the tested substrate range (2-1000 microM) and interestingly showed a biphasic kinetic profile with apparent Km,1 and Vmax,1 values of 47.2 +/- 12.5 microM and 8.1 +/- 1.4 pmol/min/pmol p450, respectively. Experiments with pooled human liver microsomes also revealed biphasic nonsaturable kinetics with apparent Km,1 and Vmax,1 values of 57.0 +/- 20.9 microM and 199.7 +/- 59.7 pmol/min/mg of protein for the high affinity enzyme, respectively. Incubation of 2 microM MPPP with 3 microM of the CYP2D6-specific inhibitor quinidine resulted in significant (p < 0.01) turnover inhibition (11.8 +/- 1.6% of control). Based on kinetic data corrected for the relative activity factors, CYP2D6 is the enzyme mainly responsible for MPPP hydroxylation, confirmed by CYP2D6 inhibition studies.     

Effect of antipsychotic drugs on human liver cytochrome P-450 (CYP) isoforms in vitro: preferential inhibition of CYP2D6.

The ability of antipsychotic drugs to inhibit the catalytic activity of five cytochrome P-450 (CYP) isoforms was compared using in vitro human liver microsomal preparations to evaluate the relative potential of these drugs to inhibit drug metabolism. The apparent kinetic parameters for enzyme inhibition were determined by nonlinear regression analysis of the data. All antipsychotic drugs tested competitively inhibited dextromethorphan O-demethylation, a selective marker for CYP2D6, in a concentration-dependent manner. Thioridazine and perphenazine were the most potent, with IC(50) values (2.7 and 1.5 microM) that were comparable to that of quinidine (0.52 microM). The estimated K(i) values for CYP2D6-catalyzing dextrorphan formation were ranked in the following order: perphenazine (0.8 microM), thioridazine (1.4 microM), chlorpromazine (6.4 microM), haloperidol (7.2 microM), fluphenazine (9.4 microM), risperidone (21.9 microM), clozapine (39.0 microM), and cis-thiothixene (65.0 microM). No remarkable inhibition of other CYP isoforms was observed except for moderate inhibition of CYP1A2-catalyzed phenacetin O-deethylation by fluphenazine (K(i) = 40.2 microM) and perphenazine (K(i) = 65.1). The estimated K(i) values for the inhibition of CYP2C9, 2C19, and 3A were >300 microM in almost all antipsychotics tested. These results suggest that antipsychotic drugs exhibit a striking selectivity for CYP2D6 compared with other CYP isoforms. This may reflect a remarkable commonality of structure between the therapeutic targets for these drugs, the transporters, and metabolic enzymes that distribute and eliminate them. Clinically, coadministration of these medicines with drugs that are primarily metabolized by CYP2D6 may result in significant drug interactions.     

Polymorphism in diazepam metabolism in Wistar rats

We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats of Wistar strain we examined showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals at the substrate concentrations less than 5 microM. We classified them as extensive metabolizer (EM) and poor metabolizer (PM) of diazepam. No sex difference was observed in the frequency of appearance of EM. Activities of the primary metabolic pathways of diazepam were examined to elucidate the cause of this polymorphism in male Wistar rats. No significant differences were observed in activities of neither diazepam 3-hydroxylation or N-desmethylation between EM and PM rats, while activity of diazepam p-hydroxylation was markedly (more than 200 times) higher in EM rats, indicating that this reaction is responsible for the polymorphism of diazepam metabolism in Wistar rats. We examined the expression levels of CYP2D1, which was reported to catalyze diazepam p-hydroxylation in Wistar rats to find no differences in the expression levels of CYP2D1 between EM and PM rats. The kinetic study on diazepam metabolism in male Wistar rats revealed that EM rats had markedly higher V(max) and smaller K(m) in diazepam p-hydroxylation than those of PM rats, indicating the presence of high affinity high capacity p-hydroxylase enzyme in EM rats. As a consequence, at low concentrations of diazepam, major pathways of diazepam metabolism were p-hydroxylation and 3-hydroxylation in male EM rats, while in male PM rats, 3-hydroxylation followed by N-desmethylation. Due to this kinetic nature of p-hydroxylase activity, EM rats had markedly higher total CL(int) of diazepam than that of PM rats. Polymorphism in diazepam metabolism in humans is well documented, but this is the first report revealing the presence of the polymorphism in diazepam metabolism in rats. The current results infer polymorphic expression of new diazepam p-hydroxylating enzyme with lower K(m) than CYP2D1 in EM Wistar rats.     

Inhibitory effects of cytostatically active 6-aminobenzo[c]phenanthridines on cytochrome P450 enzymes in human hepatic microsomes

Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(+)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 microM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.     

Role of human hepatic cytochrome P-450s in territrem A metabolism

The ability of human liver microsomal preparations (HLM1, 2, 3, and 5), microsomes from human lymphoblasts expressing different cytochrome P-450 (CYP450) isoforms, and CYP3A4 cDNA-transfected V79 Chinese hamster cells to metabolize territrem A (TRA) was studied. The only metabolite generated by any of these preparations was 6beta-hydroxymethyl-6beta-demethylterritrem A (MA(1)). MA(1) formation was observed with all four human liver microsomal samples. Of the eight microsomal preparations from human lymphoblasts expressing different cytochrome P-450 enzymes (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, only those expressing CYP2C9, CYP2D6, or CYP3A4 metabolized TRA, with that expressing CYP3A4 being the most active. No TRA metabolites were formed by control V79MZ cells, but MA(1) was formed by CYP3A4 cDNA-transfected V79 Chinese hamster cells. In order to investigate which CYP450 isoforms were involved in MA(1) formation in the human liver microsomal preparations, the effects of six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketaconazole, and diethyldithiocarbamate) and anti-3A4, anti-2C9, and anti-2D6 antibodies on TRA metabolism by HLM2 and HLM5 were examined. MA(1) formation was markedly inhibited by ketaconazole, with quinidine and sulfaphenazole having less of an effect. Anti-CYP3A4 antibody markedly inhibited MA(1) formation, while antibodies against CYP2C9 or CYP2D6 had little effect. The amount of MA(1) formed using different HLM preparations was related to the 6beta-testosterone hydroxylase activity and CYP3A4 protein content of the preparations. These results suggest that CYP3A4 is the major enzyme involved in TRA metabolism by human liver microsomes, with CYP2C9 and CYP2D6 playing a minor role.     

Genotype and phenotype of cytochrome P450 2D6 in human immunodeficiency virus-positive patients and patients with acquired immunodeficiency syndrome

Objective: To examine the distribution of the cytochrome P ₄₅₀ (CYP) CYP2D6 phenotype and its relation to genotype, concomitant medication, and disease state in human immunodeficiency virus (HIV)-positive patients. Design: A cross sectional study with a longitudinal component compared individual genotypes for CYP2D6 to the CYP2D6 phenotype. Methods: Sixty-one predominately male Caucasian, HIV-positive patients were recruited and CYP2D6 genotypes [extensive metabolizer (EM) or poor metabolizer (PM)] determined by polymerase chain reaction (PCR)-based amplification, followed by restriction fragment-length analysis. The patients were also phenotyped using dextromethorphan (DM) to determine their respective enzyme activity and assigned either a CYP2D6 EM or PM phenotype. Complete medical and treatment histories were compiled. A total of 44 patients were tested longitudinally. Results: Fifty-nine patients (97%) possessed an EM genotype, consistent with previously observed distributions in demographically similar populations. In healthy seronegative populations, genotype and phenotype have been shown to be essentially interchangeable measures of CYP2D6 activity. In this cohort, 2 of the 59 patients with an EM genotype expressed a PM phenotype. In addition, 4 EM patients were less extensive DM metabolizers than any of the patients receiving medication known to inhibit CYP2D6. This apparent shift toward the PM phenotype from the EM genotype was associated with the presence of active illness. Conclusion: Changes may occur in HIV-positive patients such that their CYP2D6 activity approaches that of PMs, despite having an EM genotype. Neither active disease nor drug interactions alone explain the shift. Received: 1 September 1999 / Accepted in revised form: 10 February 2000     

Evidence for a role of cytochrome P450 2D6 and 3A4 in ethylmorphine metabolism.

Ethylmorphine is metabolised by N-demethylation (to norethylmorphine) and by O-deethylation (to morphine). The O-deethylation reaction was previously shown in vivo to co-segregate with the O-demethylation of dextromethorphan indicating that ethylmorphine is a substrate of polymorphic cytochrome P450(CYP)2D6. To study further the features of ethylmorphine metabolism we investigated its N-demethylation and O-deethylation in human liver microsomes from eight extensive (EM) and one poor metaboliser (PM) of dextromethorphan. Whereas N-demethylation varied only two-fold there was a 4.3-fold variation in the O-deethylation of ethylmorphine, the lowest rate being observed in the PM. Quinidine, at a concentration of 1 microM, inhibited O-deethylation in microsomes from an EM, but was unable to do so in microsomes from the PM. The immunoidentified CYP2D6 and CYP3A4 correlated with the rates of O-deethylation (r = 0.972) and N-demethylation (r = 0.969), respectively. We conclude that the O-deethylation of ethylmorphine is catalysed by the CYP2D6 in human liver microsomes consistent with previous findings in healthy volunteers.